The catalytic site for ATP synthesis by mitochondrial oxidative phosphorylation is on a membrane-bound enzyme, MF1, that can be readily prepared in a soluble form. The structure mechanism, and regulation of MF1 is the subject of this proposal. MF1 contains three catalytic and three noncatalytic nucleotide binding sites. We find diadenosine-5'-polyphosphates, such as AP5A and AP6A, to inhibit F1. Inhibition requires the presence of at least one vacant noncatalytic site in addition to vacant catalytic sites; suggesting that the bifunctional ligand spans a catalytic and a noncatalytic site. To test this, we will determine the effect of AP6A binding on the number of remaining accessible catalytic and noncatalytic sites. We will also synthesize the bifunctional 2-azido analog of AP6A and use it to photolabel MF1. We ill determine whether the crosslinked sites are on the same or adjacent subunits and whether the modified residues re known to contribute to the catalytic and noncatalytic sites. Addition studies of the structure of nucleotide sites on MF1 will include determining whether structural asymmetry affects the photolabeling of residues at the noncatalytic site. We will also synthesize the 6-azido analog of ATP and attempt to identify additional regions of the nucleotide binding domains. MF1 has been reported to show triphasic kinetics in the micromolar concentration range for ATP. We plan to test the possibility that one of the breaks in the kinetic plots comes from filling a vacant noncatalytic sites. Using a hydrolysis substrate such as GTP, we will test the effect of low concentrations of 2-azido-ADP on the activity. If the rate is altered, we will photolyze and identify the site involved. We will assay for a slow transphosphorylation reaction between adjacent catalytic and noncatalytic sites that may be involved in regulation. These experiments will include testing for phosphoryl transfer between nucleotide at the catalytic site and 2-azido nucleotide that has been covalently tethered to a noncatalytic site. We will attempt to assess the effect of structural asymmetry on the mechanism of MF1 and MF0F1. We will determine whether the permanent structural asymmetry evident for noncatalytic sites on MF1 causes pre-existing functional asymmetry among the catalytic sites. We will also determine whether the unique properties of the noncatalytic sites are randomized during ATP hydrolysis by MF0F1. This approach should provide a test a notary mechanism that has been proposed.